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Plant Physiology 132:2135-2143 (2003) © 2003 American Society of Plant Biologists PAUSED Encodes the Arabidopsis Exportin-t Ortholog1Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104–6018
Los1p/exportin-t (XPOT) mediates the nuclear export of tRNAs in yeast and mammals. The requirements for this transport pathway are unclear, however, because los1 mutations do not affect yeast growth, and the phenotype of XPOT mutations in mammals is unknown. Here, we show that PAUSED (PSD) is the Arabidopsis ortholog of LOS1/XPOT and is capable of rescuing the tRNA export defect of los1 in Brewer's yeast (Saccharomyces cerevisiae), suggesting that its function has been conserved. Putative null alleles of PSD disrupt the initiation of the shoot apical meristem and delay leaf initiation after germination, the emergence of the radicle and lateral roots, and the transition to flowering. Plants doubly mutant for psd and hasty, the Arabidopsis ortholog of exportin 5, are viable but have a more severe phenotype than either single mutant. These results suggest that PSD plays a role in tRNA export in Arabidopsis, but that at least one—and perhaps several—additional tRNA export pathways also exist. The PSD transcript is broadly expressed during development and is alternatively spliced in the 3'-untranslated region. No temporal or spatial difference in the abundance of different splice forms was observed. We propose that the mutant phenotype of psd reflects defects in developmental events and cell/tissue types that require elevated levels of protein synthesis and are therefore acutely sensitive to a reduction in tRNA export.
Nucleocytoplasmic transport is mediated by a family of structurally related proteins known as karyopherins. These proteins, also called importins and exportins, bind a variety of protein and RNA cargoes and guide their movement through the nuclear pore. Many fundamental cellular processes, such as the nuclear import of ribosomal proteins and the export of tRNA molecules, require importin or exportin activity (for review, see Nakielny and Dreyfuss, 1999  ).
Karyopherins also mediate nucleocytoplasmic transport in response to
environmental or developmental cues. For example, in Brewer's yeast
(Saccharomyces cerevisiae), the karyopherins Pse1p and Msn5p regulate
the nuclear import and export of the transcription factor Pho4p in response to
phosphate levels (Kaffman et al.,
1998a,
1998b), and in fruitfly
(Drosophila melanogaster), the importin 7 ortholog, DIM-7,
and the importin- ortholog, KETEL, are required to transport
the Ser kinase D-ERK into the nucleus in response to EGFR signaling
(Lorenzen et al., 2001).
The karyopherin family has been best characterized in Brewer's yeast, where
it consists of 14 members. The loss-of-function phenotype has been determined
for all of the members of this family, and 12 have a known transport direction
and at least one identified cargo
(Costanzo et al., 2001
The Arabidopsis genome contains approximately 17 predicted karyopherins
(Bollman et al., 2003
In this paper, we describe the mutant phenotype of PAUSED
(PSD), a third member of the Arabidopsis karyopherin family.
PSD is a widely expressed gene that encodes a protein with homology
to the tRNA export receptors Los1p in Brewer's yeast and exportin-t (XPOT) in
humans. We found that PSD partially rescues the tRNA export defect of
los1, supporting the conclusion that PSD can function as an export
receptor for tRNAs. Mutant alleles of PSD (including several that are
likely to be functionally null) are viable, but have defects in a variety of
developmental processes. Null mutations of LOS1 are also viable
(Hurt et al., 1987
psd Disrupts the Onset of Shoot Growth
PSD was identified in a screen for mutations that affect meristem
initiation during embryogenesis. An initial characterization of the
psd-1 mutant phenotype demonstrated that psd
transiently disrupts the organization of the shoot apical meristem (SAM) and
delays leaf production, but does not have a significant effect on the timing
of the transition to the adult phase of vegetative development
(Telfer et al., 1997
In a Columbia (Col) background, psd mutations delay the appearance
of the first two leaves by approximately 2 d
(Fig. 1, A–D;
Table I). To determine whether
the delay in leaf emergence resulted from a failure in leaf initiation or a
decreased rate of leaf expansion, we examined leaf initiation in
psd-6 plants carrying a LFY:GUS transgene
as a marker for leaf primordia (Blazquez et
al., 1997
On average, the psd-6 plants produced their first abaxial
trichomes on leaf 2.7, whereas wild-type plants first produced abaxial
trichomes on leaf 4.9 (Fig.
1E). Despite this basipetal shift in the position of leaves with
adult characteristics, both mutant and wild-type plants produced their first
adult leaf primordium 5 DAP (Fig.
1E) and produced two transition leaves before producing leaves
with a full complement of abaxial trichomes
(Fig. 1, C and D). This
suggests that the shift in leaf identity in psd is due to the delay
in leaf production, not an alteration in the timing of the juvenile to adult
transition. This observation is consistent with previous results obtained with
psd-1 in a Landsberg erecta (Ler)
background (Telfer et al.,
1997
The delay in leaf production in psd mutants is associated with
disruptions in the anatomy of the SAM
(Telfer et al., 1997
To determine when these defects in the SAM arise, we used confocal
microscopy to examine the morphology of the SAM in mature embryos and young
seedlings. The SAM of mature psd-6 mutant seeds is greatly reduced
compared with wild type and shows no evidence of developing leaf primordia
(Fig. 2, A and B). At 1 DAP, we
observed a similar reduction in the size of the psd-6 SAM and
evidence of the loss of nuclei in the central region (data not shown). By 3
DAP, the central region of the SAM was still disrupted, but peripheral regions
were more organized, and leaf primordia were often evident
(Fig. 2, C and D). Similar
phenotypes were observed in the embryos and young seedlings of
psd-13 mutant plants (data not shown). The effect of
psd on the SAM of mature embryos indicates that the psd
phenotype arises before germination, and the loss of leaf primordia is not
specific to the juvenile phase of development. This conclusion is supported by
the fact that psd can suppress the leaf-to-cotyledon transformation
that occurs during the embryogenesis of amp1 mutant plants
(Conway and Poethig,
1997
In addition to delaying the initiation of the SAM, psd also delays
the transition from vegetative to reproductive development. Although mutant
plants have fewer juvenile and adult leaves than normal, they actually have an
increased number of leaves in the inflorescence (bracts;
Fig. 1, C and D;
Table I). Furthermore, the
first bract of mutant plants sometimes resembles an adult vegetative leaf in
having a very short internode, a slightly broader shape, and a residual
petiole (Fig. 1, C and D).
Whereas wild-type plants begin to lose adaxial trichomes at the base of the
first bract and completely lack adaxial trichomes by the third bract,
psd plants lose adaxial trichomes more gradually, and often still
have adaxial trichomes on the fifth or sixth bract. Finally,
psd-6 plants express AP1:GUS—a
marker for floral induction (Hempel et
al., 1997 psd mutations also affect the morphogenesis of the inflorescence: Phyllotaxy is disrupted in about 25% of plants, and roughly 10% of secondary inflorescences lack a subtending bract (Fig. 1, F and G). In some cases, a very small piece of leaf tissue is present beneath the secondary inflorescence, suggesting that bract production was aborted. psd also reduces fertility, with psd plants containing about 85% fewer seeds per silique than wild-type plants (Table I).
psd seeds germinate at the same frequency as wild type, but exhibit a delay in the emergence of the radicle from the seed coat, and initially produce a short, thick root (Table I). Mutant seedlings also have fewer lateral roots than normal seedlings at 14 DAP. psd does not appear to have a significant effect on the overall growth of the root, however, because there was no difference in primary root length between psd and wild-type plants at 14 DAP (Table I). To determine whether this delay in root emergence is associated with a defect in meristem organization, we examined the structure of psd-6 roots 3 DAP. In contrast to the SAM, there was no sign of cell death in the root apical meristem (RAM) of psd-6 plants: All of the cell layers were present, although cells appeared larger than wild type (Fig. 2, G and H), and there was significantly more starch in the cortex of mutant roots. It is unclear whether this phenotype reflects an effect of psd on root morphogenesis or whether the initial delay in root growth results in mutant roots being developmentally less advanced than wild-type roots. In either case, this result suggests that PSD is not required by the RAM in the same way that it is required in the SAM.
PSD maps near the marker nga111 at the bottom of chromosome 1. Additional markers from this region were generated using Ler sequence information provided by Cereon; these allowed us to delimit PSD to a region spanned by the bacterial artificial chromosome (BACs) T16D11 and T10N11. Southern analysis using T16D11 as a probe revealed an inversion in the fast neutron allele, psd-6. A 2-kb HindIII fragment disrupted by this inversion was cloned and sequenced and was found to contain a portion of a predicted open reading frame. To confirm that this breakpoint was responsible for the psd phenotype, a genomic fragment containing the predicted open reading frame plus 2.8 kb of upstream sequence, and 300 bp of downstream sequence was introduced into psd-6 and was found to restore wild-type growth and fertility (Table I).
The 2-kb HindIII fragment was used to probe a seedling cDNA
library, from which three partial clones were obtained, and the 5' and
3' ends of the transcript were isolated by RACE. The PSD
transcript totals 3.6 kb and contains 14 exons; the last two exons are
alternatively spliced, giving rise to multiple 3'-untranslated regions
(UTRs; GenBank BK001280; Fig.
3A). The PSD cDNA encodes a predicted protein of 988
amino acids, which is 27% identical to the human tRNA nuclear export factor,
XPOT (Fig. 3B). Like both yeast
and humans, Arabidopsis appears to have a single XPOT gene, because there are
no other genes in Arabidopsis that are more similar to
LOS1/XPOT than they are to another member of the
karyopherin/importin-
The DNA sequence of the other existing psd alleles revealed that all contain mutations within the same open reading frame (Fig. 3A). The EMS-induced allele psd-1 has a deletion of T2701, resulting in a premature stop codon in exon 10. One fast-neutron allele, psd-13, has a deletion of G404 and the other, psd-15, has a substitution of the sequence AATAA for the 13 bases beginning with G581. Both psd-13 and psd-15 cause premature stop codons in exon 1. We observed no difference in the phenotype of the various alleles, and given the placement of the psd-13 and psd-15 mutations, we believe that the defects described above represent the null phenotype of XPOT in Arabidopsis.
A single PSD transcript of approximately 4 kb was observed on northern blots of poly(A) RNA; this transcript was reduced in both size and abundance by the psd-6 inversion (Fig. 4A). Low levels of the PSD transcript were observed in roots, vegetative leaves, and floral buds (Fig. 4B). In addition, semiquantitative RT-PCR revealed that PSD is expressed at similar levels in the shoot apex of plants grown for 8, 15, or 22 d under short-day conditions (Fig. 4C). The widespread expression of the PSD gene is not surprising, given the pleiotropic mutant phenotype and the fundamental nature of its predicted role in tRNA export.
The sequence of several cDNAs and 3'-RACE products revealed that the 3'-UTR of PSD is alternatively spliced (Fig. 3A). Exon 13A is 9 bp shorter than exon 13B at the 3' end. Exon 14 is also alternatively spliced. Exon 14A extends uninterrupted to the polyadenylation site, whereas 14B1 and 14B2 are created by the presence of an additional intron that removes 88 bp from G3196 to G3283. We observed no correlation between the splicing pattern and the site of polyadenylation. Using RT-PCR, we examined the expression of the 3'-splicing variants, and found that 13A/14A, 13B/14A, and 13B/14B are present in the shoots of juvenile, adult, and reproductive plants, and in the roots, vegetative leaves, and floral buds of mature plants (Fig. 4C). The 13B/14A form appears to be the predominant form in all tissues, and we saw no evidence of the expression of the 13A/14B combination in the 3'-RACE products, cDNAs, or RT-PCR products. Together, the northern and RT-PCR data suggest that neither PSD expression nor the splicing of the 3'-UTR is developmentally regulated. However, we cannot exclude the possibility that PSD is differentially spliced in some regions of the tissues that were assayed by RT-PCR.
PSD is most closely related to the human XPOT gene and
its Brewer's yeast ortholog LOS1. los1 mutants are viable but lack
suppressor-tRNA activity due to defects in tRNA export
(Hopper et al., 1980
Like los1, psd mutants are viable, suggesting that Arabidopsis has
alternate mechanisms for tRNA export. Recently, the karyopherin XPO5 has also
been shown to bind tRNA (Bohnsack et al.,
2002 Plants doubly mutant for psd-1 and hst-1 show a combination of the psd and hst mutant phenotypes in that they had the accelerated trichome production and upwardly curled leaves typical of hst but lacked their first two leaves due to the psd-related delay in leaf initiation (Fig. 6). The plants also displayed a further decrease in size compared with the single-mutant parents. This same phenotype was observed for plants doubly mutant for psd-13 and hst-6, alleles that introduce stop codons near the 5' end of these genes (data not shown). The fact that double mutant plants are viable demonstrates that HST is not required for the survival of psd mutant plants. This could indicate that HST is not involved in tRNA transport or that it is not the only protein that can mediate an alternative tRNA export pathway in Arabidopsis.
The Arabidopsis psd mutation causes delays in a wide range of processes, including leaf production, root elongation, and the vegetative to reproductive transition. In keeping with the pleiotropic nature of the phenotype, we found that PSD is a widely expressed gene coding for the only Arabidopsis ortholog of LOS1/XPOT. This protein has been shown to facilitate the nuclear export of tRNA in both yeast and human systems (Arts et al., 1998a ;
Kutay et al., 1998;
Sarkar and Hopper, 1998).
Despite the low level of sequence identity, we have shown that PSD
can partially rescue the yeast los1 mutation, suggesting that the
ability to mediate tRNA export has been conserved.
Because tRNA export is an essential cellular process, it is surprising that
psd mutant plants are viable. Although it is possible that the
mutations we have identified still retain some function, we consider this
unlikely, because the psd-13 and psd-15
mutations are predicted to delete more than 85% of the protein and disrupt the
conserved N-terminal Ran-binding region. It is also unlikely that survival is
due to the presence of multiple XPOT genes, because PSD is the only
gene in the Arabidopsis genome with significant homology to XPOT
(Bollman et al., 2003
Like psd, null mutations in the single XPOT gene in Brewer's
yeast, LOS1, are viable (Hurt et
al., 1987
Recently, XPO5 has been proposed to play a role in mediating tRNA export.
The human XPO5 protein has been shown to bind directly to tRNA, and to form a
complex with eEF-1A (Bohnsack et al.,
2002
Given the general requirement for tRNAs, the effect of psd
mutations on specific developmental processes is difficult to explain. One
possibility is that PSD is temporally or spatially regulated.
Although we have shown that the PSD transcript is present in a
variety of tissues and developmental stages, we cannot rule out the
transcriptional regulation of PSD at a tissue- or cell-specific
level. It is also possible that the translation of PSD is
differentially regulated; the 3'-UTR is often the target of molecules
that regulate translation (for review, see
Gray and Wickens, 1998
The processes affected by psd might also represent developmental
events that are particularly sensitive to the loss of the XPOT pathway.
Although psd mutations affect many aspects of Arabidopsis
development, they have particularly significant effects on major developmental
transitions, including the initiation of the SAM, radicle emergence, lateral
root initiation, and the transition to flowering. These events may
specifically require the XPOT pathway or may require the simultaneous function
of all tRNA export pathways to meet translational demands. It is also
important to consider the potential for interplay between potential tRNA
export pathways. Although XPOT shows a high specificity for tRNA
(Arts et al., 1998a
Plant Growth and Culture
Arabidopsis plants of the ecotype Col-0 were grown under constant light at
22°C. To examine root morphology, plants were grown under constant light
on 1x Murashige and Skoog salts (Invitrogen, Carlsbad, CA), 2.5
mM MES, 4.5% (w/v) Suc, and 0.9% (w/v) agar
(Malamy and Benfey, 1997
psd-1 was isolated by ethyl methane sulfonate mutagenesis
of Ler as described previously
(Barton and Poethig, 1993
psd-6 plants were crossed to Col plants carrying
LFY::GUS (gift of D. Weigel) or AP1::GUS
(gift of M. Yanofsky).
To visualize nuclei, seedlings were fixed overnight in a 1:1 (v/v) mixture
of hepatane:fix (80 mM EGTA, 5% [v/v] formaldehyde, and 0.1% [v/v]
Tween 20 in phosphate-buffered saline) and cleared in 95% (v/v) ethanol. They
were then incubated in 70:30 (v/v) ethanol:PBT-0.1 (phosphate-buffered saline
and 0.1% [v/v] Tween 20), 50:50 (v/v) ethanol:PBT-0.1, and stained for 10 min
in 1 g mL-1 Hoechst 33258 (Sigma-Aldrich, St. Louis) in PBT-0.1.
After three washes in PBT-0.1, the seedlings were mounted in Aqua-polymount
(Polysciences, Warrington, PA) and imaged using a confocal microscope (TCS-NT,
Leica, Wetzlar, Germany). Histological analysis was carried out on seedlings
embedded in Spurr's resin, as described previously
(Conway and Poethig,
1997
PSD was mapped near nga111 on the bottom of chromosome 1, using an F2 mapping population of 343 plants from a cross between psd-1(Ler) and wild-type Col. BACs from this region were hybridized to genomic DNA from the psd-6 allele, and a HindIII polymorphism was detected using BAC F28P22 as a probe. The corresponding 2-kb HindIII fragment from Col was isolated and used to probe a seedling cDNA library (gift of J. Ecker). Four partial cDNAs corresponding to At1g72560 were isolated and sequenced (GenBank BK001280), and the ends were isolated using the 5'- and 3'-RACE systems (Invitrogen). To locate the mutations in the psd-1, psd-13, and psd-15 alleles, reverse transcription was carried out using Superscript II (Invitrogen), and PCR products were generated using the primers: PSD1B (5'-GTATGCAACAAACCAGGACTGG-3'); PSD5862 (5'-CTCTCCGATGAAGATTCAACAGC-3'); PSD5758 (5'-AGATCTCTTTGTCTTATTACG-3'); PSD583 (5'-ATCATGGAGAGCGCTGTTGAA-3'); PSD7431 (5'-CCCAAGGCTTTGGAACAGCTAC-3'); and PSD9417 (5'-CATCGACTGCAGAGAGTTATAAG-3'). The products of these reactions were gel isolated using the QIAquick gel extraction kit (Qiagen USA, Valencia, CA) before sequencing.
A 7.6-kb SalI-PstI genomic fragment containing the
PSD predicted open reading frame plus 2.8 kb of upstream sequence and
300 bp of downstream sequence was isolated from the BAC T16D11 and subcloned
into the SalI and PstI sites of pCAMBIA2300 (CAMBIA). As a
negative control, a BamHI fragment containing 4.4 kb of upstream DNA
and the first 1 kb of the PSD open reading frame was subcloned into
the HindIII site of pCAMBIA2300. Both constructs were transformed
into Agrobacterium tumefaciens strain GV3101 pMP90 by
electroporation. psd-6 mutant plants were transformed by the
floral dip method (Clough and Bent,
1998
Leaf and floral bud tissues were isolated from mature plants, and root
tissue was grown in culture (see above). Eight-, 15-, and 22-d shoot apices
were obtained by removing the roots and all visible leaves from the shoots of
Col plants grown under short-day conditions (8 h of light and 16 h of dark).
RNA was prepared using TRIzol reagent (Invitrogen), and poly(A) RNA was
isolated using the Oligotex mRNA kit (Qiagen USA). Poly(A) RNA (0.75 µg)
was run on a 1.2% (w/v) agarose gel containing 3% (v/v) formaldehyde and was
transferred to Hybond N+ membrane (Amersham Biosciences, Uppsala). Probes to
PSD were generated by combining RT-PCR products amplified with the
PSD1B-PSD1C (5'-TAGCCAAAACCTGTGCAAGC-3'), PSD5758-PSD583, and
PSD7431-PSD9417 primer pairs. Actin probes were generated using the primers
5'-AAGATGACCCAAATCATGTTTGAGAC-3' and
5'-ACGACCTTGATCTTCATGCTGC-3'. Probes were labeled using the
Prime-it II kit (Stratagene, La Jolla, CA), and hybridization was carried out
as described by Smith et al.
(1992
For analysis of the expression of alternatively spliced transcripts, RNA
from the above tissues was reverse transcribed, and PCR was carried out for
10, 15, 20, or 25 cycles using a combination of primers PSD8947
(5'-GACTAGTGTCGACCGGGAGTCACGTTTTCAGATAG-3') and PSD9417 at a 1
µM concentration, and the primers ACT8F
(5'-GCCAGATCTTCATCGTCGTGGTTCTTG-3') and ACT8R
(5'-CCAATGGCGTAAAGTGATAGAACAGC-3') at a 0.125 µM
concentration. The resulting products were digested with DdeI to
distinguish the 13A and 13B forms, and the digests were run on a 4% (w/v)
MetaPhor gel (BMA Biomedicals, Augst, Switzerland) and transferred to Hybond
N+ membrane (Amersham Biosciences). Probes were generated using the same
primer sets, and hybridization was carried out by standard methods
(Sambrook et al., 1989
A 3.5-kb HpaI-SalI fragment containing the PSD
open reading frame was ligated to the SmaI and SalI sites of
the p426ADH vector (ATCC). The ligation mixture was transformed into the
Brewer's yeast strain X2316 (MAT
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We are grateful to Randy Kerstetter, Tanya Berardini, and Matt Sauer for advice and helpful discussions and to Kathy Tworkowski for technical assistance. We also thank Joe Ecker, Anita Hopper, Marty Yanofsky, and Detlef Weigel for reagents used in this study. Received March 10, 2003; returned for revision April 6, 2003; accepted May 6, 2003.
1 This work was supported by the National Institutes of Health (grants to C.A.H. and R.S.P.). 
2 Present address: DuPont Ag Biotech, Delaware Technology Park, Suite 200, 1
Innovation Way, Newark, DE 19711. * Corresponding author; e-mail spoethig{at}sas.upenn.edu; fax 215–898–8780.
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ABSTRACT
RESULTS
SUP4 his5-2 lys1-1
can1-100 trp5-48 ade2-1 ura3-1; gift of A. Hopper) transformants were
selected on synthetic dextrose complete (SDC) (-ura) plates and were screened
for the insertion using the PSD-specific primers PSD469
(5'-GTCTTTACTGCTTACCCCTCT-3') and PSD583. Plasmid DNA was isolated
from selected transformants and was used to transform the strain 201-1-5
(MAT
